The present invention relates to derivatives of 4xe2x80x2-hydroxyazobenzene-2-carboxylic acid (HABA) and to intermediates therefor, and to their use as novel reagents for labeling, isolation and detection of biological molecules.
There are two major systems currently in use for the nonradioactive labeling of proteins and other biologically active molecules. These are the avidin-biotin system and the DIG (digoxygenin) system. In both cases, a reactive reagent, containing either the biotin or DIG moiety, is usually used for covalent coupling to a binding molecule, e.g. DNA, protein, etc, which in turn will recognize a target molecule. Once biotin or DIG is thus incorporated into the experimental system, avidin or DIG-specific antibody (anti-DIG) is applied subsequently to serve as a bridge between the target and a probe that is required for a desired purpose such as for detection, localization, quantification, isolation, etc.
Both avidin-biotin and the DIG systems suffer from a series of disadvantages. One of the main problems of the avidin-biotin system is that biotin is a natural vitamin, and as such, it is a normal component of cells either in the free state or covalently bound to a group of biotin-dependent enzymes. Thus, the presence of free biotin will interfere with the targeting of avidin to a biotinylated molecule, whereas the presence of biotin-dependent enzymes will result in specific but unwanted binding of avidin. A second disadvantage with the avidin-biotin system is that in some cases a reversible binding between avidin and biotin would be desirable, whereas the extremely high affinity essentially results in an irreversible binding. Another problem involves the analysis and processing of a biotinylated preparation. Specifically, since biotin is not chromogenic, it is difficult to quantify the number of biotin moieties per molecule and, more relevant, to determine the percentage of molecules which failed to undergo biotinylation. In this context, it is not easy to separate between biotinylated and non-biotinylated molecules, after the biotinylation step.
The DIG:anti-DIG system also suffers from a set of disadvantages. First, it has been noted that DIG reacts nonspecifically with antibodies from serum. Like biotin, the lack of chromophore renders it difficult to determine the amount of DIG-labeled molecules and to separate them from the unlabeled fraction.
Both systems suffer from another major drawback in that only one type of protein is available for detection, i.e. avidin or streptavidin for binding biotin and anti-DIG for DIG.
It is an object of the present invention to provide new labeling reagents based on derivatives of the azo dye HABA (4xe2x80x2-hydroxyazobenzene-2-carboxylic acid).
The present invention relates to compounds of the formula I: 
wherein
R is H or xe2x80x94Nxe2x95x90N-2-carboxyphenyl;
A is (CH2)n or xe2x80x94CHxe2x95x90CHxe2x80x94, wherein n is an integer from 0 to 10, or
A may also be xe2x80x94CH(COOH)xe2x80x94 when R is xe2x80x94Nxe2x95x90N-2-carboxyphenyl; and
X is a radical selected from the group consisting of:
(a) Cl;
(b) COOR1, wherein R1 is p-nitrophenyl or N-succinimidyl;
(c) CONHxe2x80x94NHR2, wherein R2 is H, COO(t-butyl) or COObenzyl;
(d) CONHxe2x80x94[B]xe2x80x94NHR3, wherein R3 is H, COOR1, or COxe2x80x94[Bxe2x80x2]-maleimido, wherein R1 is t-butyl, p-nitrophenyl or N-succinimidyl, and B and Bxe2x80x2, the same or different, are (CH2)n wherein n is an integer from 2 to 10;
(e) CONHxe2x80x94[B]xe2x80x94COOR4, wherein B is as defined in (iv) above and R4 is H, C1-C8 alkyl, N-succinimidyl;
(f) CONHxe2x80x94[B]xe2x80x94OH, wherein B is as defined in (iv) above;
(g) CONHxe2x80x94[B]xe2x80x94CONHxe2x80x94NHR2, wherein B is as defined in (iv) above and R2 is H, COO(t-butyl) or COObenzyl; and
(h) NHR2, wherein R2 is H, COO(t-butyl) or COObenzyl, when A is xe2x80x94CH(COOH)xe2x80x94 and R is xe2x80x94Nxe2x95x90N-2-carboxyphenyl.
The HABA compounds of formula I, wherein R is xe2x80x94Nxe2x95x90N-2-carboxyphenyl, are obtained from the corresponding non-azo compounds wherein R is H.
In one embodiment, A is (CH2)n and n is 2 to 4, preferably 2, and the compounds wherein R is H are derivatives of 3-(2-hydroxyphenyl)-propionic acid (derivatives (ii) above),-propionamide (derivatives (iv) to (vii) above) and propionic acid hydrazide (derivatives (iii) above). In another embodiments, B is preferably (CH2)5 or (CH2)6 and Bxe2x80x2 is preferably (CH2)3.
The present invention further relates to conjugates of HABA itself or of a HABA compound of formula I with a carrier (herein HABAylated compounds), wherein said carrier is a protein or polypeptide, an amino-carrying polymer, a polynucleotide, an oligonucleotide, a polysaccharide, an oligosaccharide or a compound containing a sugar molecule such as glycocoproteins. Thus the invention encompasses HABAylated cytokines, antibodies, hormones, receptors, DNA, DNA probes, oligonucleotides and other HABAylated molecules.
In another aspect, the present invention provides anti-HABA antibodies, both polyclonal and monoclonal, which are prepared by immunization of rabbits and mice, respectively, with a conjugate of HABA and an immunogenic protein, such as for example HABA-KLH.
The HABA derivatives of formula I can be used as labeling reagents. Labeled molecules are amenable to interaction with either avidin or HABA-specific antibodies (anti-HABA) and can be used in several applications including, but not being limited to, isolation (affinity chromatography) and detection (immunoassay) of biologically active molecules.
The invention further provides a method for localization, quantitation and isolation of molecules I in a sample which comprises contacting the sample with a HABAylated molecule II that recognizes molecule I, and then reacting with labeled anti-HABA antibodies or labeled avidin. The HABAylated molecule II such as an HABAylated antibody, lectin, DNA or RNA, is added to a sample such as a cell preparation, a DNA or protein blot containing the target molecule II, e.g. the antigen, the carbohydrate, etc, the excess of HABAylated probe is removed after formation of the HABAylated binder II/target molecule I complex and then reacted with labeled anti-HABA antibodies or labeled avidin.
The HABA system has many advantages over both the avidin-biotin and the DIG/anti-DIG systems, as follows:
HABA can be detected by two unrelated systems: avidin and anti-HABA. Interestingly, the interaction between HABA and anti-HABA generates a spectral shift similar to that of the HABA-avidin interaction from 350 to 500 nm. Anti-HABA fails to recognize biotin.
The HABA moiety is easily detectable and quantifiable, owing to its inherent chromophore and spectral shift upon binding to avidin or anti-HABA.
It is easy to separate between HABAylated and non-HABAylated molecules, due to the reversible interaction with avidin.
HABAylated molecules can be recognized by avidin and, after addition of biotin, the HABAylated molecule is then again available for detection by anti-HABA antibodies.